Whole-transcriptomic effects of bentonite, when used to prevent aflatoxin B1 toxicity, on a widely used in vitro model of intestinal epithelium

Introduction: Aflatoxin B1 (AFB1) is a major food and feed safety concern. Strategies to prevent aflatoxicosis are limiting the mycotoxin production in the field, decreasing its presence in food and feed commodities and, recently, using phytocompounds as feed additives. Bentonite (BEN) is a clay widely used to reduce AFB1 presence in contaminated feedstuffs; however, few studies fully characterized its toxicity. We showed that BEN concentrations up to 0.1 mg/mL did not affect cell viability and permeability and reduced AFB1 cytotoxicity. In this study we assessed the whole-transcriptomic effects (RNA-seq) of BEN, AFB1 and their combination (AFB1+BEN) in Caco-2 cells. Materials and Methods: Cells were seeded and left grown for 21 days. To increase CYP3A4 expression and activity, cells were pre-treated for 24 hrs with 12-O-tetradecanoylphorbol 13-acetate and sodium butyrate. Monolayers were incubated for 48 hrs with 81 μM AFB1, 0.1 mg/mL BEN or their combination. Controls (CTRL) were represented by cells incubated with 0.25% dimethylsulfoxide. Total RNA was isolated (Qiagen RNeasy mini kit); after its concentration and quality assessment, tagged libraries were prepared using the Illumina TruSeq Stranded mRNA kit and sequenced on an Illumina NextSeq 500 instrument using a 75 bp single end approach. Reads were counted, quality checked and trimmed using validated pipelines. Reads were mapped to Homo sapiens reference genome (STAR software). EdgeR and clusterProfiler software were used to identify differentially expressed genes (DEGs) and carry out the functional enrichment analysis, respectively. Results: We obtained more than 34 million raw reads/sample and a mean mapping percentage of 88%. In the MultiDimensional Scale Plot, AFB1 clustered away from CTRL and BEN samples, that were closed to each other. Interestingly, AFB1+BEN samples clustered in between of aforementioned clusters. Pair-wise comparisons of BEN vs CTRL, AFB1 vs CTRL and AFB1+BEN vs AFB1 identified 0, 7005, and 2198 DEGs, respectively. When analyzing AFB1 vs CTRL and AFB1+BEN vs AFB1, we identified 2018 common DEGs, and 2008 showed an opposite behavior, suggesting a protective role of BEN toward AFB1. Pathways for which BEN showed a protective effect were cell integrity, xenobiotic metabolism and transporters, basal metabolism, inflammation and immune response, p53 biological network, apoptosis and carcinogenesis. Conclusions: This is the first in vitro study assessing whole-transcriptomic effects of BEN, alone or in combination with AFB1. BEN did not affect Caco-2 cells transcriptome. AFB1 provoked most of transcriptional changes. However, BEN was preventive.